Inbred sunflower SA425A

ABSTRACT

The present invention relates to an inbred sunflower, designated SA425A. The invention relates to the seeds of inbred sunflower SA425A, to the plants of inbred sunflower SA425A and to methods for producing a sunflower plant by crossing cultivar SA425A with itself or another sunflower. The invention further relates to methods for producing a sunflower plant containing in its genetic material one or more transgenes and to the transgenic sunflower plants and plant parts produced by those methods and to methods for producing other hybrid sunflower derived from inbred sunflower SA425A.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority from U.S. provisionalpatent application Ser. No. 62/145,640 filed on Apr. 10, 2015, which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The present invention relates to a new sunflower inbred (Helianthusannuus L.) designated SA425A. All publications cited in this applicationare herein incorporated by reference.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possess the traits to meetthe program goals. The goal is to combine in a single variety or hybridan improved combination of desirable traits from the parental germplasm.These important traits may include increased head size and weight,higher seed yield, improved color, resistance to diseases and insects,tolerance to drought and heat, and better agronomic quality.

The cultivated sunflower (Helianthus annuus L.) is a major worldwidesource of vegetable oil. In the United States, the major sunflowerproducing states are the Dakotas, Minnesota, Kansas, Colorado, Nebraska,Texas and California, although most states have some commercial acreage.

Sunflowers are considered oilseeds, along with cottonseed, soybeans andcanola and the growth of sunflower as an oilseed crop has rivaled thatof soybean. The oil accounts for 80% of the value of the sunflower crop,as contrasted with soybean which derives most of its value from themeal. Sunflower oil is generally considered a premium oil because of itslight color, high level of unsaturated fatty acids, lack of linolenicacid, bland flavor and high smoke points. The primary fatty acids in theoil are oleic and linoleic with the remainder consisting of palmitic andstearic saturated fatty acids.

Non-dehulled or partly dehulled sunflower meal has been substitutedsuccessfully for soybean meal in isonitrogenous (equal protein) dietsfor ruminant animals, as well as for swine and poultry feeding.Sunflower meal is higher in fiber, has a lower energy value and is lowerin lysine but higher in methionine than soybean meal. Protein percentageof sunflower meal ranges from 28% for non-dehulled seeds to 42% forcompletely dehulled seeds.

In addition to its use in food and food products for humans and animals,sunflower oil also has industrial uses. It has been used in paints,varnishes and plastics because of good semidrying properties without thecolor modification associated with oils high in linolenic acid. It hasalso been used in the manufacture of soaps, detergents and cosmetics.The use of sunflower oil (and other vegetable oils) as a pesticidecarrier, and in the production of agrichemicals, surfactants, adhesives,fabric softeners, lubricants and coatings has been explored.Considerable work has also been done to explore the potential ofsunflower as an alternate fuel source in diesel engines becausesunflower oil contains 93% of the energy of US Number 2 diesel fuel(octane rating of 37). Sunflower oil has also been proposed as a sourceof hydrogen for hydrogen fuel cells. (BBC News, Aug. 26, 2004).

Sunflower is an annual, erect, broadleaf plant with a strong taproot anda prolific lateral spread of surface roots. Stems are usually roundearly in the season, angular and woody later in the season, and normallyunbranched. The sunflower head is not a single flower (as the nameimplies) but is made up of 1,000 to 2,000 individual flowers joined at acommon receptacle. The flowers around the circumference are ligulate rayflowers without stamens or pistils; the remaining flowers are perfectflowers with stamens and pistils. Anthesis (pollen shedding) begins atthe periphery and proceeds to the center of the head. Since manysunflower varieties have a degree of self-incompatibility, pollenmovement between plants by insects is important, and bee colonies havegenerally increased yields.

There are two basic types of sunflowers grown in North America: 1)oil-type sunflower used for oilseed production and 2) non-oilseed, orconfectionery sunflower, used for food and birdseed. Oilseed sunflowerseeds are usually smaller and black in color, whereas non-oilseed orconfectionery sunflower seeds are larger than oilseed and are black andwhite striped.

At least 30 diseases, caused by various fungi, bacteria and viruses,have been identified on wild or cultivated sunflower, but only a few areof economic significance as far as causing yield losses. The sunflowerdiseases Phoma Black Stem (Phoma macdonaldii), Phomopsis Stem Canker(Phomopsis helianthi), Verticillium Leaf Mottle (Verticillium dahliae)and Sclerotinia (Scerotinia sclerotiorum) are some of the mostsignificant disease problems in sunflower producing areas of China,United States and Europe.

The development of a cytoplasmic male-sterile and restorer system forsunflower has enabled seed companies to produce high-quality hybridseed. Most of these have higher yields than open-pollinated varietiesand are higher in percent oil. Performance of varieties tested overseveral environments is the best basis for selecting sunflower hybrids.The choice should consider yield, oil percentage, maturity, seed size(for non-oilseed markets), and lodging and disease resistance.

Therefore, it is desirable to develop new sunflower varieties havingresistance to significant sunflower diseases while also producing highseed yields with excellent seed characteristics, such as novel seedcolor.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described inconjunction with systems, tools, and methods which are meant to beexemplary and illustrative, not limiting in scope. In variousembodiments, one or more of the above-described problems have beenreduced or eliminated, while other embodiments are directed to otherimprovements.

According to the invention, there is provided a novel inbred sunflowerdesignated SA425A. This invention thus relates to the seeds of inbredsunflower SA425A, to the plants of inbred sunflower SA425A and tomethods for producing a sunflower plant by crossing sunflower SA425Awith itself or another sunflower plant, to methods for producing asunflower plant containing in its genetic material one or moretransgenes and to the transgenic sunflower plants produced by thatmethod. The invention also relates to hybrid plants and seeds and anyfurther progeny or descendants of the inbred derived by crossing inbredsunflower SA425A as a pollen donor. Thus, any methods using inbredsunflower SA425A in backcrosses, hybrid production, crosses topopulations, and the like, are part of this invention. All plants whichare a progeny of or descend from inbred sunflower SA425A are within thescope of this invention. It is an aspect of this invention for inbredsunflower SA425A to be used in crosses with other, different, sunflowerplants to produce first generation (F₁) sunflower hybrid seeds andplants with superior characteristics.

Another aspect of the invention is to provide methods for producingother sunflower plants derived from inbred sunflower SA425A. Sunflowerplants derived by the use of those methods are also part of theinvention.

In another aspect, the present invention provides for single gene ormultiple gene converted plants of inbred sunflower SA425A. The single ormultiple transferred gene(s) may preferably be a dominant or recessiveallele. Preferably, the single or multiple transferred gene(s) willconfer such traits as herbicide resistance, insect resistance,resistance to bacterial, fungal, or viral diseases, male fertility, malesterility, enhanced nutritional quality, and industrial usage. Thesingle or multiple gene(s) may be a naturally occurring sunflower geneor a transgene introduced through genetic engineering techniques.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of inbred sunflower SA425A. The tissue culturewill preferably be capable of regenerating plants having thephysiological and morphological characteristics of the foregoingsunflower plants, and of regenerating plants having substantially thesame genotype as the foregoing sunflower plants. Genetic variants ofinbred sunflower SA425A naturally generated through the use of tissueculture or artificially induced utilizing mutagenic agents during tissueculture are aspects of the present invention. Preferably, theregenerable cells in such tissue cultures will be embryos, protoplasts,meristematic cells, callus, pollen, cotyledon, leaves, flowers, anthers,roots, pistils, root tips, glumes, seeds, panicles or stems. Stillfurther, the present invention provides sunflower plants regeneratedfrom the tissue cultures of the invention.

The invention further provides methods for developing sunflower plantsand sunflower hybrid plants in a sunflower plant breeding program usingplant breeding techniques including recurrent selection, backcrossing,pedigree breeding, restriction fragment length polymorphism enhancedselection, genetic marker enhanced selection, and transformation. Seeds,sunflower plants, and parts thereof, produced by such breeding methodsare also part of the invention.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference bystudy of the following descriptions.

DETAILED DESCRIPTION OF THE INVENTION

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Abiotic stress. As used herein, abiotic stress relates to all non-livingchemical and physical factors in the environment. Examples of abioticstress include, but are not limited to, drought, flooding, salinity,temperature, and climate change.

Allele. The allele is any of one or more alternative forms of a gene,all of which relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotype of the F₁ hybrid.

Cell. Cell as used herein includes a plant cell, whether isolated, intissue culture or incorporated in a plant or plant part.

Cotyledon. One of the first leaves of the embryo of a seed plant;typically one or more in monocotyledons, two in dicotyledons, and two ormore in gymnosperms.

Dominant trait or dominant allele. As used herein in regards to mutantallele NSMA, means dominant or partially dominant and expressed in manygenetic backgrounds.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics of the recurrent parent, except for the characteristicsderived from the converted gene.

F_(#). The “F” symbol denotes the filial generation, and the # is thegeneration number, such as F₁, F₂, F₃, etc.

Gene. As used herein, “gene” refers to a segment of nucleic acid. A genecan be introduced into a genome of a species, whether from a differentspecies or from the same species, using transformation or variousbreeding methods.

Genetically Modified. Describes an organism that has received geneticmaterial from another, or had its genetic material modified, resultingin a change in one or more of its phenotypic characteristics. Methodsused to modify, introduce or delete the genetic material may includemutation breeding, backcross conversion, genetic transformation, singleand multiple gene conversion, and/or direct gene transfer.

Genotype. Refers to the genetic constitution of a cell or organism.

Length/Width (L/W) Ratio. This ratio is determined by dividing theaverage length (L) by the average width (W).

Linkage. Refers to a phenomenon wherein alleles on the same chromosometend to segregate together more often than expected by chance if theirtransmission was independent.

Linkage Disequilibrium. Refers to a phenomenon wherein alleles tend toremain together in linkage groups when segregating from parents tooffspring, with a greater frequency than expected from their individualfrequencies.

Locus. A locus confers one or more traits such as, for example, malesterility, herbicide tolerance, insect resistance, disease resistance,waxy starch, modified fatty acid metabolism, modified phytic acidmetabolism, modified carbohydrate metabolism and modified proteinmetabolism. The trait may be, for example, conferred by a naturallyoccurring gene introduced into the genome of the variety bybackcrossing, a natural or induced mutation, or a transgene introducedthrough genetic transformation techniques. A locus may comprise one ormore alleles integrated at a single chromosomal location.

Marker-assisted selection (MAS). Also called marker-assisted breeding.The use of DNA markers that are tightly-linked to target loci as asubstitute for or to assist phenotypic screening. Ideally, the markerused for selection associates at high frequency with the gene orquantitative trait locus of interest, due to genetic linkage. Markerloci that are tightly linked to major genes can be used for selectionand are sometimes more efficient than direct selection for the targetgene.

Multiple Gene Converted (Conversion). Multiple gene converted(conversion) includes plants developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of an inbred are recovered, whileretaining two or more genes transferred into the inbred via crossing andbackcrossing. The term can also refer to the introduction of multiplegenes through genetic engineering techniques known in the art.

NSMA. Refers to the novel mutant allele that confers white seed colorand is found in inbred sunflower SA425A, as well as in numerous othersunflower developed by the inventors. NSMA appears to be a dominantmutant allele. The white seed color gene NSMA was identified as a singlegene that underlies the seed color regulation and was mapped to aninterval of 5.2 centimorgan (cM) on the long arm of linkage group LG16in sunflower and was tightly linked to flanking SNP markers NHA006022and NHA006025.

Percent Identity. Percent identity as used herein refers to thecomparison of the homozygous alleles of two sunflower varieties. Percentidentity is determined by comparing a statistically significant numberof the homozygous alleles of two developed varieties. For example, apercent identity of 90% between sunflower variety 1 and sunflowervariety 2 means that the two varieties have the same allele at 90% oftheir loci.

Percent Similarity. Percent similarity as used herein refers to thecomparison of the homozygous alleles of a sunflower variety with anothersunflower plant, and if the homozygous allele of both sunflower plantsmatches at least one of the alleles from the other plant then they arescored as similar. Percent similarity is determined by comparing astatistically significant number of loci and recording the number ofloci with similar alleles as a percentage. A percent similarity of 90%between the sunflower plant of this invention and another plant meansthat the sunflower plant of this invention matches at least one of thealleles of the other sunflower plant at 90% of the loci.

Plant. As used herein, the term “plant” includes reference to animmature or mature whole plant, including a plant from which seed orgrain or anthers have been removed. Seed or embryo that will produce theplant is also considered to be the plant.

Plant Height. Plant height in centimeters is taken from soil surface tothe tip at harvest.

Plant Parts. As used herein, the term “plant parts” (or a soybean plant,or a part thereof) includes but is not limited to protoplasts, leaves,stems, roots, root tips, anthers, pistils, seed, grain, embryo, pollen,ovules, cotyledon, hypocotyl, pod, flower, shoot, tissue, petiole,cells, meristematic cells, and the like.

Pubescence. This refers to a covering of very fine hairs closelyarranged on the leaves, stems and glumes of the plant.

Quantitative Trait Loci. Quantitative Trait Loci (QTL) refers to geneticloci that control to some degree, numerically representable traits thatare usually continuously distributed.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

RHS. RHS refers to the Royal Horticultural Society of England whichpublishes an official botanical color chart quantitatively identifyingcolors according to a defined numbering system. The chart may bepurchased from Royal Horticulture Society Enterprise Ltd., RHS Garden;Wisley, Woking; Surrey GU236QB, UK.

Rogueing. Rogueing is the process in seed production where undesiredplants are removed from a variety. The plants are removed since theydiffer physically from the general desired expressed characteristics ofthe variety. The differences can be related to size, color, maturity,leaf texture, leaf margins, growth habit, or any other characteristicthat distinguishes the plant.

Single gene converted. Single gene converted or conversion plant refersto plants which are developed by a plant breeding technique calledbackcrossing or via genetic engineering wherein essentially all of thedesired morphological and physiological characteristics of a line arerecovered in addition to the single gene transferred into the line viathe backcrossing technique or via genetic engineering.

SNP. Refers to single nucleotide polymorphisms. Variation at a singleposition in a DNA sequence among individuals; SNPs are usuallyconsidered to be point mutations that have been evolutionarilysuccessful enough to recur in a significant proportion of the populationof a species. If an SNP occurs within a gene, then the gene is describedas having more than one allele. SNPs can also occur in noncoding regionsof DNA. If certain SNPs are known to be associated with a trait, thenstretches of DNA near the SNPs can be examined in an attempt to identifythe gene or genes responsible for the trait.

Sunflower. Helianthus annuus L., as used herein, sunflower includesconfectionery, oilseed (oil-type) and conoil sunflower types. Conoilsunflower refers to sunflower hybrids developed using both oil-type andconfection parentage. Conoils tend to have higher oil content thantraditional confection varieties.

White-seeded or white seed color. As used herein in describing thepresent invention, refers to the white sunflower seed color conferred bymutant allele NSMA, which is found in inbred sunflower SA425A as well asnumerous other hybrid combinations using SA425A as a parent. The whiteseed color includes, but is not limited to, the following RHS colorsdepending on the genetic background and environment: RHS 155A-D,N155A-D, 156A-D, 157A-D, 158A-D, and 159A-D.

The present invention of sunflower inbred SA425A is a cytoplasmic malesterile sunflower line having CMS PET1 cytoplasm derived from H.petiolaris (French). Sunflower inbred SA425A contains a novel dominantmutant allele designated NSMA, which confers white seed color. Themaintainer line of SA425A is designated SA425B. The mutant allele NSMAof the present invention is readily transferred to other sunflowerplants and the white seed color conferred by NSMA is present in allhybrid combinations tested using SA425A as a parent. Mutant allele NSMAappears to be a novel gene conferring dominant white hull color, whichis surprising because seed color is typically controlled by severalgenes. When inbred sunflower SA425A is used in crosses, the resultinghybrid sunflowers have white seed color.

Additionally, sunflower inbred SA425A is a confectionary sunflower typeand is resistant to imidazolinone herbicides.

Inbred sunflower SA425A can be compared to public line HA292. Inbredsunflower SA425A is taller than HA292. Additionally, inbred sunflowerSA425A is resistant to imidazolinone herbicides, whereas HA292 issusceptible to imidazolinone herbicides.

The inbred has shown uniformity and stability, as described in thefollowing variety description information. It has been self-pollinated asufficient number of generations with careful attention to uniformity ofplant type. The line has been increased with continued observation foruniformity.

Inbred sunflower SA425A has the following morphological andphysiological characteristics (based primarily on data collected inBreckenridge, Minn.). This description is in accordance with UPOVterminology.

TABLE 1 VARIETY DESCRIPTION INFORMATION Classification: Family:Asteraceae Botanical name: Helianthus annuus L. Common name: SunflowerMaturity: Days to stage R5.1 (from emergence): 67 Days to physiologicalmaturity (from emergence): 109 Plant: Natural height (at M0 stage):Tall; 127.0 cm Branching (excluding environmental branching, at M0-M2stage): Absent Hypocotyl (at A2 stage): Anthocyanin coloration: AbsentLeaves (at E4 stage): Size: Large Blade length (mid-stem at flowering):25.0 cm Number of leaves: 24 Green color: Medium Blistering: MediumSerration: Medium Shape: Cordate Shape of cross section: Weakly concaveShape of distal part: Lanceolate Apex: Acuminate Base: AuriculateMargin: Medium serrate Margin color: Green Depth of margin indentations:Intermediate Attitude: Descending Surface: Crinkled (ridged) Auricules:Small Wings: Weakly expressed Angle of lowest lateral veins: AcuteHeight of the tip of the blade compared to insertion of petiole (at 2/3height of plants): Medium Stem (at F1 stage): Length of internodes atharvest ripeness: 15.0 cm Hairiness at the top (last 5 cm): Medium Timeof flowering: Medium Ray florets (at F3.2 stage): Density: Medium Shape:Narrow ovate Disposition: Longitudinal recurved Length: Long; 28.0 mmWidth: 13.0 mm Color: Medium yellow Appearance: Flat Disk flower (atF3.2 stage): Color: Yellow Anthocyanin coloration of stigma: AbsentProduction of pollen: Absent Bract (at F3.2 stage): Shape: Clearlyelongated Length of tip: Medium Green color of outer side: MediumAttitude in relation to head at M0 stage: Not embracing or very slightlyembracing Head (at M3 stage): Attitude: Turned down with strongly curvedstem Size: Large Diameter: 27.0 cm Shape of grain side: Flat Seeds (atM4 stage): Size: Very large Length: 17.0 mm Weight (gm/100 seeds): 11.5gm Percent held on 7.9 mm (20/64) round-hole scree: 59% Shape: Narrowovoid Shape in cross section: Not curved Thickness relative to width:Thick Main color (outer pericarp): White Middle pericarp: White Innerpericarp: No color Stripes on margin: None or very weakly expressedStripes between margins: None or very weakly expressed Spots onpericarp: Absent Oil data:  Hull: 30%  Oil: 21%  Fatty acids (expressedas percentages of oil):   Oleic: 26%   Linoleic: 64%   Palmitic: 3.1%  Stearic: 6.9% Resistance to pests/diseases/herbicides: Downy mildew(Plasmopara halstedii): Susceptible Banded sunflower moth (Cochylishospes): Susceptible Seed weevil (Smicronyx fulvus or S. sordidus):Susceptible Sunflower beetle (Zygogramma exclamationis): SusceptibleImidazolinone: Resistant

This invention is also directed to methods for producing a sunflowerplant by crossing a first parent sunflower plant with a second parentsunflower plant, wherein the first parent sunflower plant or secondparent sunflower plant is the sunflower plant from inbred sunflowerSA425A. Further, both the first parent sunflower plant and second parentsunflower plant may be from inbred sunflower SA425A. Therefore, anymethods using inbred sunflower SA425A are part of this invention:selfing, backcrosses, hybrid breeding, and crosses to populations. Anyplants produced using inbred sunflower SA425A as at least one parent arewithin the scope of this invention.

Still further, this invention also is directed to methods for producinga hybrid sunflower SA425A-derived sunflower plant by crossing inbredsunflower SA425A with a second sunflower plant and growing the progenyseed, and repeating the crossing and growing steps with inbred sunflowerSA425A-derived plant from 0 to 7 times. Thus, any such methods using theinbred sunflower SA425A are part of this invention: selfing,backcrosses, hybrid production, crosses to populations, and the like.All plants produced using inbred sunflower SA425A as a parent are withinthe scope of this invention, including plants derived from inbredsunflower SA425A.

Further Embodiments of the Invention

Sunflower is an important and valuable vegetable crop. Thus, acontinuing goal of sunflower plant breeders is to develop stable, highyielding hybrid sunflowers that are agronomically sound. To accomplishthis goal, the sunflower breeder must select and develop sunflowerplants with traits that result in superior cultivars.

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts, as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach hasbeen used extensively for breeding disease-resistant cultivars. Variousrecurrent selection techniques are used to improve quantitativelyinherited traits controlled by numerous genes. The use of recurrentselection in self-pollinating crops depends on the ease of pollination,the frequency of successful hybrids from each pollination, and thenumber of hybrid offspring from each successful cross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard, theoverall value of the advanced breeding lines, and the number ofsuccessful cultivars produced per unit of input (e.g., per year, perdollar expended, etc.).

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for at least three years. The best lines are candidatesfor new commercial cultivars. Those still deficient in a few traits areused as parents to produce new populations for further selection.

These processes, which lead to the final step of marketing anddistribution, usually take from ten to twenty years from the time thefirst cross or selection is made. Therefore, development of newcultivars is a time-consuming process that requires precise forwardplanning, efficient use of resources, and a minimum of changes indirection.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of sunflower plant breeding is to develop new, unique, andsuperior hybrid sunflowers. The breeder initially selects and crossestwo or more parental lines, followed by repeated selfing and selection,producing many new genetic combinations. The breeder can theoreticallygenerate billions of different genetic combinations via crossing,selfing, and mutations. The breeder has no direct control at thecellular level. Therefore, two breeders will never develop the sameline, or even very similar lines, having the same sunflower traits.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under different geographical,climatic, and soil conditions, and further selections are then madeduring, and at the end of, the growing season. The cultivars that aredeveloped are unpredictable. This unpredictability is because thebreeder's selection occurs in unique environments, with no control atthe DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.The same breeder cannot produce the same line twice by using the exactsame original parents and the same selection techniques. Thisunpredictability results in the expenditure of large research monies todevelop superior hybrid sunflowers.

The development of commercial hybrid sunflowers requires the developmentof sunflower varieties, the crossing of these varieties, and theevaluation of the crosses. Pedigree breeding and recurrent selectionbreeding methods are used to develop cultivars from breedingpopulations. Breeding programs combine desirable traits from two or morevarieties or various broad-based sources into breeding pools from whichcultivars are developed by selfing and selection of desired phenotypes.The new cultivars are crossed with other varieties and the hybrids fromthese crosses are evaluated to determine which have commercialpotential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents which possess favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁'s or by intercrossing two F₁'s (sib mating). Selection of the bestindividuals is usually begun in the F₂ population. Then, beginning inthe F₃, the best individuals in the best families are selected.Replicated testing of families, or hybrid combinations involvingindividuals of these families, often follows in the F₄ generation toimprove the effectiveness of selection for traits with low heritability.At an advanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror line that is the recurrent parent. The source of the trait to betransferred is called the donor parent. The resulting plant is expectedto have the attributes of the recurrent parent (e.g., cultivar) and thedesirable trait transferred from the donor parent. After the initialcross, individuals possessing the phenotype of the donor parent areselected and repeatedly crossed (backcrossed) to the recurrent parent.The resulting plant is expected to have the attributes of the recurrentparent (e.g., cultivar) and the desirable trait transferred from thedonor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineswith each generation due to failure of some seeds to germinate or someplants to produce at least one seed. As a result, not all of the F₂plants originally sampled in the population will be represented by aprogeny when generation advance is completed.

In addition to phenotypic observations, the genotype of a plant can alsobe examined. There are many laboratory-based techniques available forthe analysis, comparison and characterization of plant genotype; amongthese are Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length polymorphisms (AFLPs), Simple Sequence Repeats(SSRs—which are also referred to as Microsatellites), and SingleNucleotide Polymorphisms (SNPs).

Isozyme Electrophoresis and RFLPs have been widely used to determinegenetic composition. Shoemaker and Olsen (Molecular Linkage Map ofSoybean (Glycine max), pp. 6.131-6.138 in S. J. O'Brien (ed.) GeneticMaps: Locus Maps of Complex Genomes, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1993)) developed a molecular geneticlinkage map that consisted of 25 linkage groups with about 365 RFLP, 11RAPD, three classical markers, and four isozyme loci. See also,Shoemaker, R. C., RFLP Map of Soybean, pp. 299-309, in Phillips, R. L.and Vasil, I. K. (eds.), DNA-Based Markers in Plants, Kluwer AcademicPress, Dordrecht, the Netherlands (1994).

SSR technology is currently the most efficient and practical markertechnology; more marker loci can be routinely used and more alleles permarker locus can be found using SSRs in comparison to RFLPs. Forexample, Diwan and Cregan described a highly polymorphic microsatellitelocus in soybean with as many as 26 alleles. Diwan, N. and Cregan, P.B., Theor. Appl. Genet., 95:22-225 (1997). SNPs may also be used toidentify the unique genetic composition of the invention and progenyvarieties retaining that unique genetic composition. Various molecularmarker techniques may be used in combination to enhance overallresolution.

Molecular markers, which include markers identified through the use oftechniques such as Isozyme Electrophoresis, RFLPs, RAPDs, AP-PCR, DAF,SCARs, AFLPs, SSRs, and SNPs, may be used in plant breeding. One use ofmolecular markers is Quantitative Trait Loci (QTL) mapping. QTL mappingis the use of markers which are known to be closely linked to allelesthat have measurable effects on a quantitative trait. Selection in thebreeding process is based upon the accumulation of markers linked to thepositive effecting alleles and/or the elimination of the markers linkedto the negative effecting alleles from the plant's genome.

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers can also beused to select toward the genome of the recurrent parent and against themarkers of the donor parent. This procedure attempts to minimize theamount of genome from the donor parent that remains in the selectedplants. It can also be used to reduce the number of crosses back to therecurrent parent needed in a backcrossing program. The use of molecularmarkers in the selection process is often called genetic marker enhancedselection or marker-assisted selection. Molecular markers may also beused to identify and exclude certain sources of germplasm as parentalvarieties or ancestors of a plant by providing a means of trackinggenetic profiles through crosses.

Mutation breeding is another method of introducing new traits intosunflower varieties. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means including temperature, long-term seed storage,tissue culture conditions, radiation (such as X-rays, Gamma rays,neutrons, Beta radiation, or ultraviolet radiation), chemical mutagens(such as base analogs like 5-bromo-uracil), antibiotics, alkylatingagents (such as sulfur mustards, nitrogen mustards, epoxides,ethyleneamines, sulfates, sulfonates, sulfones, or lactones), azide,hydroxylamine, nitrous acid, or acridines. Once a desired trait isobserved through mutagenesis the trait may then be incorporated intoexisting germplasm by traditional breeding techniques. Details ofmutation breeding can be found in Principles of Cultivar Development byFehr, Macmillan Publishing Company (1993).

The production of double haploids can also be used for the developmentof homozygous varieties in a breeding program. Double haploids areproduced by the doubling of a set of chromosomes from a heterozygousplant to produce a completely homozygous individual. For example, seeWan, et al., Theor. Appl. Genet., 77:889-892 (1989).

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Principles of Plant Breeding, John Wiley and Son, pp.115-161 (1960); Allard (1960); Simmonds (1979); Sneep, et al. (1979);Fehr (1987); “Carrots and Related Vegetable Umbelliferae,” Rubatzky, V.E., et al. (1999).

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Any DNA sequences,whether from a different species or from the same species, which areintroduced into the genome using transformation or various breedingmethods, are referred to herein collectively as “transgenes.” Over thelast fifteen to twenty years, several methods for producing transgenicplants have been developed, and the present invention, in particularembodiments, also relates to transformed versions of the claimed line.

Nucleic acids or polynucleotides refer to RNA or DNA that is linear orbranched, single or double stranded, or a hybrid thereof. The term alsoencompasses RNA/DNA hybrids. These terms also encompass untranslatedsequence located at both the 3′ and 5′ ends of the coding region of thegene: at least about 1000 nucleotides of sequence upstream from the 5′end of the coding region and at least about 200 nucleotides of sequencedownstream from the 3′ end of the coding region of the gene. Less commonbases, such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine,and others can also be used for antisense, dsRNA, and ribozyme pairing.For example, polynucleotides that contain C-5 propyne analogues ofuridine and cytidine have been shown to bind RNA with high affinity andto be potent antisense inhibitors of gene expression. Othermodifications, such as modification to the phosphodiester backbone, orthe 2′-hydroxy in the ribose sugar group of the RNA can also be made.The antisense polynucleotides and ribozymes can consist entirely ofribonucleotides, or can contain mixed ribonucleotides anddeoxyribonucleotides. The polynucleotides of the invention may beproduced by any means, including genomic preparations, cDNApreparations, in vitro synthesis, RT-PCR, and in vitro or in vivotranscription.

Plant transformation involves the construction of an expression vectorthat will function in plant cells. Such a vector comprises DNAcomprising a gene under control of, or operatively linked to, aregulatory element (for example, a promoter). The expression vector maycontain one or more such operably linked gene/regulatory elementcombinations. The vector(s) may be in the form of a plasmid, and can beused alone or in combination with other plasmids, to provide transformedsunflower plants using transformation methods as described below toincorporate transgenes into the genetic material of the sunflowerplant(s).

Expression Vectors for Sunflower Transformation: Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (for example, a promoter) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or an herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from transposonTn5, which when placed under the control of plant regulatory signalsconfers resistance to kanamycin. Fraley, et al., PNAS, 80:4803 (1983).Another commonly used selectable marker gene is the hygromycinphosphotransferase gene which confers resistance to the antibiotichygromycin. Vanden Elzen, et al., Plant Mol. Biol., 5:299 (1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant. Hayford, et al., Plant Physiol.,86:1216 (1988); Jones, et al., Mol. Gen. Genet., 210:86 (1987); Svab, etal., Plant Mol. Biol., 14:197 (1990); Hille, et al., Plant Mol. Biol.,7:171 (1986). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate, or bromoxynil. Comai, etal., Nature, 317:741-744 (1985); Gordon-Kamm, et al., Plant Cell,2:603-618 (1990); and Stalker, et al., Science, 242:419-423 (1988).

Selectable marker genes for plant transformation that are not ofbacterial origin include, for example, mouse dihydrofolate reductase,plant 5-enolpyruvylshikimate-3-phosphate synthase, and plantacetolactate synthase. Eichholtz, et al., Somatic Cell Mol. Genet.,13:67 (1987); Shah, et al., Science, 233:478 (1986); and Charest, etal., Plant Cell Rep., 8:643 (1990).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include α-glucuronidase (GUS),α-galactosidase, luciferase and chloramphenicol, acetyltransferase.Jefferson, R. A., Plant Mol. Biol., 5:387 (1987); Teeri, et al., EMBOJ., 8:343 (1989); Koncz, et al., PNAS, 84:131 (1987); and DeBlock, etal., EMBO J., 3:1681 (1984).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissues are available. Molecular Probes,Publication 2908, IMAGENE GREEN, pp. 1-4 (1993) and Naleway, et al., J.Cell Biol., 115:151a (1991). However, these in vivo methods forvisualizing GUS activity have not proven useful for recovery oftransformed cells because of low sensitivity, high fluorescentbackgrounds, and limitations associated with the use of luciferase genesas selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie, et al., Science, 263:802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Expression Vectors for Sunflower Transformation: Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element (for example, a promoter).Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred.”Promoters which initiate transcription only in certain tissue arereferred to as “tissue-specific.” A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters:

An inducible promoter is operably linked to a gene for expression insunflower. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in sunflower. With an inducible promoter, therate of transcription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward,et al., Plant Mol. Biol., 22:361-366 (1993). Exemplary induciblepromoters include, but are not limited to, that from the ACEI systemwhich responds to copper (Meft, et al., PNAS, 90:4567-4571 (1993)); In2gene from maize which responds to benzenesulfonamide herbicide safeners(Hershey, et al., Mol. Gen. Genet., 227:229-237 (1991) and Gatz, et al.,Mol. Gen. Genet., 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz, etal., Mol. Gen. Genet., 227:229-237 (1991)). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena,et al., PNAS, 88:0421 (1991).

B. Constitutive Promoters:

A constitutive promoter is operably linked to a gene for expression insunflower or the constitutive promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in sunflower.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell, et al., Nature, 313:810-812 (1985)) and the promoters from suchgenes as rice actin (McElroy, et al., Plant Cell, 2:163-171 (1990));ubiquitin (Christensen, et al., Plant Mol. Biol., 12:619-632 (1989) andChristensen, et al., Plant Mol. Biol., 18:675-689 (1992)); pEMU (Last,et al., Theor. Appl. Genet., 81:581-588 (1991)); MAS (Velten, et al.,EMBO J., 3:2723-2730 (1984)) and maize H3 histone (Lepetit, et al., Mol.Gen. Genet., 231:276-285 (1992) and Atanassova, et al., Plant J., 2(3):291-300 (1992)). The ALS promoter, Xbal/Ncol fragment 5′ to theBrassica napus ALS3 structural gene (or a nucleotide sequence similarityto said Xbal/Ncol fragment), represents a particularly usefulconstitutive promoter. See PCT Application No. WO 96/30530.

C. Tissue-Specific or Tissue-Preferred Promoters:

A tissue-specific promoter is operably linked to a gene for expressionin sunflower. Optionally, the tissue-specific promoter is operablylinked to a nucleotide sequence encoding a signal sequence which isoperably linked to a gene for expression in sunflower. Plantstransformed with a gene of interest operably linked to a tissue-specificpromoter produce the protein product of the transgene exclusively, orpreferentially, in a specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai, et al., Science, 23:476-482(1983) and Sengupta-Gopalan, et al., PNAS, 82:3320-3324 (1985)); aleaf-specific and light-induced promoter such as that from cab orrubisco (Simpson, et al., EMBO J., 4(11):2723-2729 (1985) and Timko, etal., Nature, 318:579-582 (1985)); an anther-specific promoter such asthat from LAT52 (Twell, et al., Mol. Gen. Genet., 217:240-245 (1989)); apollen-specific promoter such as that from Zm13 (Guerrero, et al., Mol.Gen. Genet., 244:161-168 (1993)) or a microspore-preferred promoter suchas that from apg (Twell, et al., Sex. Plant Reprod., 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall, ormitochondrion, or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample, Becker, et al., Plant Mol. Biol., 20:49 (1992); Close, P. S.,Master's Thesis, Iowa State University (1993); Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley,” Plant Mol. Biol., 9:3-17 (1987); Lerner, et al., PlantPhysiol., 91:124-129 (1989); Fontes, et al., Plant Cell, 3:483-496(1991); Matsuoka, et al., PNAS, 88:834 (1991); Gould, et al., J. Cell.Biol., 108:1657 (1989); Creissen, et al., Plant J., 2:129 (1991);Kalderon, et al., A short amino acid sequence able to specify nuclearlocation, Cell, 39:499-509 (1984); and Steifel, et al., Expression of amaize cell wall hydroxyproline-rich glycoprotein gene in early leaf androot vascular differentiation, Plant Cell, 2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem., 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is sunflower. In anotherpreferred embodiment, the biomass of interest is seed. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRFLP, PCR, and SSR analysis, which identifies the approximatechromosomal location of the integrated DNA molecule. For exemplarymethodologies in this regard, see Methods in Plant Molecular Biology andBiotechnology, Glick and Thompson Eds., 269:284, CRC Press, Boca Raton(1993). Map information concerning chromosomal location is useful forproprietary protection of a subject transgenic plant. If unauthorizedpropagation is undertaken and crosses made with other germplasm, the mapof the integration region can be compared to similar maps for suspectplants, to determine if the latter have a common parentage with thesubject plant. Map comparisons would involve hybridizations, RFLP, PCR,SSR, and sequencing, all of which are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

A. Genes that Confer Resistance to Pests or Disease and that Encode:

1. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant line can be transformed with a clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example, Jones, et al., Science, 266:789(1994) (cloning of the tomato Cf-9 gene for resistance to Cladosporiumfulvum); Martin, et al., Science, 262:1432 (1993) (tomato Pto gene forresistance to Pseudomonas syringae pv. tomato encodes a protein kinase);and Mindrinos, et al., Cell, 78:1089 (1994) (Arabidopsis RSP2 gene forresistance to Pseudomonas syringae).

2. A Bacillus thuringiensis protein, a derivative thereof, or asynthetic polypeptide modeled thereon. See, for example, Geiser, et al.,Gene, 48:109 (1986), who disclose the cloning and nucleotide sequence ofa Bt δ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxingenes can be purchased from American Type Culture Collection, Manassas,Va., for example, under ATCC Accession Nos. 40098, 67136, 31995, and31998.

3. A lectin. See, for example, the disclosure by Van Damme, et al.,Plant Mol. Biol., 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

4. A vitamin-binding protein such as avidin. See PCT Application No. US93/06487, the contents of which are hereby incorporated by reference.The application teaches the use of avidin and avidin homologues aslarvicides against insect pests.

5. An enzyme inhibitor, for example, a protease or proteinase inhibitor,or an amylase inhibitor. See, for example, Abe, et al., J. Biol. Chem.,262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor); Huub, et al., Plant Mol. Biol., 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I); and Sumitani,et al., Biosci. Biotech. Biochem., 57:1243 (1993) (nucleotide sequenceof Streptomyces nitrosporeus α-amylase inhibitor).

6. An insect-specific hormone or pheromone, such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock, et al., Nature, 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

7. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem., 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor) and Pratt, etal., Biochem. Biophys. Res. Comm., 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also, U.S. Pat. No. 5,266,317 toTomalski, et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

8. An insect-specific venom produced in nature, by a snake, a wasp, etc.For example, see Pang, et al., Gene, 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

9. An enzyme responsible for a hyper-accumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative,or another non-protein molecule with insecticidal activity.

10. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase, and a glucanase, whether natural or synthetic. See PCTApplication No. WO 93/02197 in the name of Scott, et al., whichdiscloses the nucleotide sequence of a callase gene. DNA molecules whichcontain chitinase-encoding sequences can be obtained, for example, fromthe ATCC under Accession Nos. 39637 and 67152. See also, Kramer, et al.,Insect Biochem. Mol. Biol., 23:691 (1993), who teach the nucleotidesequence of a cDNA encoding tobacco hornworm chitinase, and Kawalleck,et al., Plant Mol. Biol., 21:673 (1993), who provide the nucleotidesequence of the parsley ubi4-2 polyubiquitin gene.

11. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella, et al., Plant Mol. Biol., 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess,et al., Plant Physiol., 104:1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

12. A hydrophobic moment peptide. See PCT Application No. WO 95/16776(disclosure of peptide derivatives of tachyplesin which inhibit fungalplant pathogens) and PCT Application No. WO 95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

13. A membrane permease, a channel former, or a channel blocker. Forexample, see the disclosure of Jaynes, et al., Plant Sci., 89:43 (1993),of heterologous expression of a cecropin-β, lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

14. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy, et al., Ann. Rev. Phytopathol.,28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus, and tobacco mosaic virus. Id.

15. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. SeeTaylor, et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions, Edinburgh, Scotland (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

16. A virus-specific antibody. See, for example, Tavladoraki, et al.,Nature, 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

17. A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo-α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient released by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb, et al., Bio/technology,10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubart,et al., Plant J., 2:367 (1992).

18. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann, et al., Bio/technology, 10:305 (1992), have shownthat transgenic plants expressing the barley ribosome-inactivating genehave an increased resistance to fungal disease.

Any of the above listed disease or pest resistance genes (1-18) can beintroduced into the claimed sunflowers through a variety of meansincluding but not limited to transformation and crossing.

B. Genes that Confer Resistance to an Herbicide:

1. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee, etal., EMBO J., 7:1241 (1988) and Miki, et al., Theor. Appl. Genet.,80:449 (1990), respectively.

2. Glyphosate (resistance conferred by mutant5-enolpyruvlshikimate-3-phosphate synthase (EPSPS) and aroA genes,respectively) and other phosphono compounds, such as glufosinate(phosphinothricin acetyl transferase (PAT), dicamba and Streptomyceshygroscopicus phosphinothricin-acetyl transferase PAT bar genes), andpyridinoxy or phenoxy proprionic acids and cyclohexones (ACCaseinhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 toShah, et al., which discloses the nucleotide sequence of a form of EPSPSwhich can confer glyphosate resistance. A DNA molecule encoding a mutantaroA gene can be obtained under ATCC Accession No. 39256, and thenucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061 to Comai. See also, Umaballava-Mobapathie in TransgenicResearch, 8:1, 33-44 (1999) that discloses Lactuca sativa resistant toglufosinate. European Patent Application No. 0 333 033 to Kumada, etal., and U.S. Pat. No. 4,975,374 to Goodman, et al., disclose nucleotidesequences of glutamine synthetase genes which confer resistance toherbicides, such as L-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in EuropeanApplication No. 0 242 246 to Leemans, et al. DeGreef, et al.,Bio/technology, 7:61 (1989), describe the production of transgenicplants that express chimeric bar genes coding for phosphinothricinacetyl transferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cyclohexones, such as sethoxydim andhaloxyfop, are the Accl-S1, Accl-S2, and Accl-S3 genes described byMarshall, et al., Theor. Appl. Genet., 83:435 (1992).

3. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibilla, et al.,Plant Cell, 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes, et al., Biochem. J.,285:173 (1992).

4. Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants. See Hattori, et al., Mol. Gen.Genet., 246:419 (1995). Other genes that confer tolerance to herbicidesinclude a gene encoding a chimeric protein of rat cytochrome P4507A1 andyeast NADPH-cytochrome P450 oxidoreductase (Shiota, et al., PlantPhysiol., 106:17 (1994)), genes for glutathione reductase and superoxidedismutase (Aono, et al., Plant Cell Physiol., 36:1687 (1995)), and genesfor various phosphotransferases (Datta, et al., Plant Mol. Biol., 20:619(1992)).

5. Protoporphyrinogen oxidase (protox) is necessary for the productionof chlorophyll, which is necessary for all plant survival. The protoxenzyme serves as the target for a variety of herbicidal compounds. Theseherbicides also inhibit growth of all the different species of plantspresent, causing their total destruction. The development of plantscontaining altered protox activity which are resistant to theseherbicides are described in U.S. Pat. Nos. 6,288,306, 6,282,837,5,767,373, and International Publication WO 01/12825.

Any of the above listed herbicide genes (1-5) can be introduced into theclaimed sunflowers through a variety of means including, but not limitedto, transformation and crossing.

C. Genes that Confer or Contribute to a Value-Added Trait, Such as:

1. Increased iron content of the sunflower, for example, by introducinginto a plant a soybean ferritin gene as described in Goto, et al., ActaHorticulturae., 521, 101-109 (2000).

2. Decreased nitrate content of leaves, for example, by introducing intoa sunflower a gene coding for a nitrate reductase. See, for example,Curtis, et al., Plant Cell Rep., 18:11, 889-896 (1999).

3. Modified fatty acid metabolism, for example, by introducing into aplant an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon, et al., PNAS, 89:2625 (1992).

4. Modified carbohydrate composition effected, for example, byintroducing into plants a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza, et al., J. Bacteriol., 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene); Steinmetz, et al., Mol. Gen. Genet., 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene);Pen, et al., Bio/technology, 10:292 (1992) (production of transgenicplants that express Bacillus lichenifonnis α-amylase); Elliot, et al.,Plant Mol. Biol., 21:515 (1993) (nucleotide sequences of tomatoinvertase genes); Søgaard, et al., J. Biol. Chem., 268:22480 (1993)(site-directed mutagenesis of barley α-amylase gene); and Fisher, etal., Plant Physiol., 102:1045 (1993) (maize endosperm starch branchingenzyme II).

D. Genes that Control Male-Sterility:

1. Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN—Ac-PPT. See International Publication WO 01/29237.

2. Introduction of various stamen-specific promoters. See InternationalPublications WO 92/13956 and WO 92/13957.

3. Introduction of the barnase and the barstar genes. See Paul, et al.,Plant Mol. Biol., 19:611-622 (1992).

For additional examples of nuclear male and female sterility systems andgenes, see also, U.S. Pat. Nos. 5,859,341, 6,297,426, 5,478,369,5,824,524, 5,850,014 and 6,265,640. See also Hanson, Maureen R., et.al., (2004) “Interactions of Mitochondrial and Nuclear Genes That AffectMale Gametophyte Development” Plant Cell. 16:S154-S169, all of which arehereby incorporated by reference.

Methods for Sunflower Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki, et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glickand Thompson Eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993). Inaddition, expression vectors and in vitro culture methods for plant cellor tissue transformation and regeneration of plants are available. See,for example, Gruber, et al., “Vectors for Plant Transformation” inMethods in Plant Molecular Biology and Biotechnology, Glick and ThompsonEds., CRC Press, Inc., Boca Raton, pp. 89-119 (1993).

A. Agrobacterium-Mediated Transformation:

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch, et al., Science, 227:1229 (1985); Curtis, et al., Journal ofExperimental Botany, 45:279, 1441-1449 (1994); Tones, et al., Plant CellTissue and Organ Culture, 34:3, 279-285 (1993); and Dinant, et al.,Molecular Breeding, 3:1, 75-86 (1997). A. tumefaciens and A. rhizogenesare plant pathogenic soil bacteria which genetically transform plantcells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes,respectively, carry genes responsible for genetic transformation of theplant. See, for example, Kado, C. I., Crit. Rev. Plant Sci., 10:1(1991). Descriptions of Agrobacterium vector systems and methods forAgrobacterium-mediated gene transfer are provided by Gruber, et al.,supra, Miki, et al., supra, and Moloney, et al., Plant Cell Rep., 8:238(1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7, 1997.

B. Direct Gene Transfer:

Several methods of plant transformation collectively referred to asdirect gene transfer have been developed as an alternative toAgrobacterium-mediated transformation. A generally applicable method ofplant transformation is microprojectile-mediated transformation whereinDNA is carried on the surface of microprojectiles measuring 1 μm to 4μm. The expression vector is introduced into plant tissues with abiolistic device that accelerates the microprojectiles to speeds of 300m/s to 600 m/s which is sufficient to penetrate plant cell walls andmembranes. Russell, D. R., et al., Plant Cell Rep., 12 (3, January),165-169 (1993); Aragao, F. J. L., et al., Plant Mol. Biol., 20 (2,October), 357-359 (1992); Aragao, F. J. L., et al., Plant Cell Rep., 12(9, July), 483-490 (1993); Aragao, Theor. Appl. Genet., 93:142-150(1996); Kim, J., Minamikawa, T., Plant Sci., 117:131-138 (1996);Sanford, et al., Part. Sci. Technol., 5:27 (1987); Sanford, J. C.,Trends Biotech., 6:299 (1988); Klein, et al., Bio/technology, 6:559-563(1988); Sanford, J. C., Physiol. Plant, 7:206 (1990); Klein, et al.,Bio/technology, 10:268 (1992).

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang, et al., Bio/technology, 9:996 (1991).Alternatively, liposome and spheroplast X4237 have been used tointroduce expression vectors into plants. Deshayes, et al., EMBO J.,4:2731 (1985) and Christou, et al., PNAS, 84:3962 (1987). Direct uptakeof DNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol, orpoly-L-ornithine has also been reported. Hain, et al., Mol. Gen. Genet.,199:161 (1985) and Draper, et al., Plant Cell Physiol., 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Saker, M., Kuhne, T., Biologia Plantarum, 40(4):507-514(1997/98); Donn, et al., In Abstracts of VIIth International Congress onPlant Cell and Tissue Culture IAPTC, A2-38, p. 53 (1990); D'Halluin, etal., Plant Cell, 4:1495-1505 (1992); and Spencer, et al., Plant Mol.Biol., 24:51-61 (1994). See also Chupean, et al., Bio/technology, 7:5,503-508 (1989).

Additional methods include, but are not limited to, expression vectorsintroduced into plant tissues using a direct gene transfer method, suchas microprojectile-mediated delivery, DNA injection, electroporation,and the like. More preferably, expression vectors are introduced intoplant tissues by using either microprojectile-mediated delivery with abiolistic device or by using Agrobacterium-mediated transformation.Transformed plants obtained with the protoplasm of the invention areintended to be within the scope of this invention.

Following transformation of sunflower target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic line. The transgenic line could then be crossedwith another (non-transformed or transformed) line in order to produce anew transgenic sunflower line. Alternatively, a genetic trait which hasbeen engineered into a particular sunflower using the foregoingtransformation techniques could be introduced into another line usingtraditional backcrossing techniques that are well known in the plantbreeding arts. For example, a backcrossing approach could be used tomove an engineered trait from a public, non-elite inbred line into anelite inbred line, or from an inbred line containing a foreign gene inits genome into an inbred line or lines which do not contain that gene.As used herein, “crossing” can refer to a simple X by Y cross, or theprocess of backcrossing, depending on the context.

Gene Conversions

When the term “sunflower plant” is used in the context of the presentinvention, this also includes any gene conversions of that variety. Theterm “gene converted plant” as used herein refers to those sunflowerplants which are developed by backcrossing, genetic engineering, ormutation, wherein essentially all of the desired morphological andphysiological characteristics of a variety are recovered in addition tothe one or more genes transferred into the variety via the backcrossingtechnique, genetic engineering, or mutation. Backcrossing methods can beused with the present invention to improve or introduce a characteristicinto the variety. The term “backcrossing” as used herein refers to therepeated crossing of a hybrid progeny back to the recurrent parent,i.e., backcrossing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more times to therecurrent parent. The parental sunflower plant which contributes thegene for the desired characteristic is termed the “nonrecurrent” or“donor parent.” This terminology refers to the fact that thenonrecurrent parent is used one time in the backcross protocol andtherefore does not recur. The parental sunflower plant to which the geneor genes from the nonrecurrent parent are transferred is known as therecurrent parent as it is used for several rounds in the backcrossingprotocol. Poehlman & Sleper (1994) and Fehr (1993). In a typicalbackcross protocol, the original variety of interest (recurrent parent)is crossed to a second variety (nonrecurrent parent) that carries thegene of interest to be transferred. The resulting progeny from thiscross are then crossed again to the recurrent parent and the process isrepeated until a sunflower plant is obtained wherein essentially all ofthe desired morphological and physiological characteristics of therecurrent parent are recovered in the converted plant, in addition tothe transferred gene from the nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a trait or characteristic in the original line.To accomplish this, a gene of the recurrent cultivar is modified orsubstituted with the desired gene from the nonrecurrent parent, whileretaining essentially all of the rest of the desired genetic, andtherefore the desired physiological and morphological characteristics ofthe original line. The choice of the particular nonrecurrent parent willdepend on the purpose of the backcross. One of the major purposes is toadd some commercially desirable, agronomically important trait to theplant. The exact backcrossing protocol will depend on the characteristicor trait being altered to determine an appropriate testing protocol.Although backcrossing methods are simplified when the characteristicbeing transferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

Many gene traits have been identified that are not regularly selected inthe development of a new line but that can be improved by backcrossingtechniques. Gene traits may or may not be transgenic. Examples of thesetraits include, but are not limited to, male sterility, modified fattyacid metabolism, modified carbohydrate metabolism, herbicide resistance,resistance for bacterial, fungal, or viral disease, insect resistance,enhanced nutritional quality, industrial usage, yield stability, andyield enhancement. These genes are generally inherited through thenucleus. Several of these gene traits are described in U.S. Pat. Nos.5,777,196, 5,948,957, and 5,969,212, the disclosures of which arespecifically hereby incorporated by reference.

Tissue Culture

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of sunflower andregeneration of plants therefrom is well known and widely published. Forexample, reference may be had to Teng, et al., HortScience, 27:9,1030-1032 (1992); Teng, et al., HortScience, 28:6, 669-1671 (1993);Zhang, et al., Journal of Genetics and Breeding, 46:3, 287-290 (1992);Webb, et al., Plant Cell Tissue and Organ Culture, 38:1, 77-79 (1994);Curtis, et al., Journal of Experimental Botany, 45:279, 1441-1449(1994); Nagata, et al., Journal for the American Society forHorticultural Science, 125:6, 669-672 (2000); and Ibrahim, et al., PlantCell Tissue and Organ Culture, 28(2), 139-145 (1992). It is clear fromthe literature that the state of the art is such that these methods ofobtaining plants are routinely used and have a high rate of success.Thus, another aspect of this invention is to provide cells which upongrowth and differentiation produce sunflower plants having thephysiological and morphological characteristics of variety SA425A orhybrid X4237.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, meristematic cells, andplant cells that can generate tissue culture that are intact in plantsor parts of plants, such as leaves, pollen, embryos, roots, root tips,anthers, pistils, flowers, seeds, petioles, suckers, and the like. Meansfor preparing and maintaining plant tissue culture are well known in theart. By way of example, a tissue culture comprising organs has been usedto produce regenerated plants. U.S. Pat. Nos. 5,959,185, 5,973,234, and5,977,445 describe certain techniques, the disclosures of which areincorporated herein by reference.

Additional Breeding Methods

This invention also is directed to methods for producing a sunflowerplant by crossing a first parent sunflower plant with a second parentsunflower plant wherein the first or second parent sunflower plant is asunflower plant of inbred sunflower SA425A. Further, both first andsecond parent sunflower plants can come from inbred sunflower SA425A.Thus, any such methods using inbred sunflower SA425A are part of thisinvention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using inbred sunflowerSA425A as at least one parent are within the scope of this invention,including those developed from cultivars derived from inbred sunflowerSA425A. Advantageously, this hybrid sunflower could be used in crosseswith other, different, sunflower plants to produce the first generation(F₁) sunflower hybrid seeds and plants with superior characteristics.The cultivar of the invention can also be used for transformation whereexogenous genes are introduced and expressed by the cultivar of theinvention. Genetic variants created either through traditional breedingmethods using inbred sunflower SA425A or through transformation ofinbred sunflower SA425A by any of a number of protocols known to thoseof skill in the art are intended to be within the scope of thisinvention.

The following describes breeding methods that may be used with inbredsunflower SA425A in the development of further sunflower plants. Onesuch embodiment is a method for developing inbred sunflower SA425Aprogeny sunflower plants in a sunflower plant breeding programcomprising: obtaining the sunflower plant, or a part thereof, of inbredsunflower SA425A, utilizing said plant or plant part as a source ofbreeding material, and selecting a inbred sunflower SA425A progeny plantwith molecular markers in common with inbred sunflower SA425A and/orwith morphological and/or physiological characteristics selected fromthe characteristics listed in Table 1. Breeding steps that may be usedin the sunflower plant breeding program include pedigree breeding,backcrossing, mutation breeding, and recurrent selection. In conjunctionwith these steps, techniques such as RFLP-enhanced selection, geneticmarker enhanced selection (for example, SSR markers), and the making ofdouble haploids may be utilized.

Another method involves producing a population of inbred sunflowerSA425A progeny sunflower plants, comprising crossing inbred sunflowerSA425A with another sunflower plant, thereby producing a population ofsunflower plants, which, on average, derive 50% of their alleles frominbred sunflower SA425A. A plant of this population may be selected andrepeatedly selfed or sibbed with a hybrid sunflower resulting from thesesuccessive filial generations. One embodiment of this invention is thehybrid sunflower produced by this method and that has obtained at least50% of its alleles from inbred sunflower SA425A.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see Fehr and Walt, Principles of CultivarDevelopment, pp. 261-286 (1987). Thus the invention includes inbredsunflower SA425A progeny sunflower plants comprising a combination of atleast two inbred sunflower SA425A traits selected from the groupconsisting of those listed in Table 1, so that said progeny sunflowerplant is not significantly different for said traits than inbredsunflower SA425A as determined at the 5% significance level when grownin the same environmental conditions. Using techniques described herein,molecular markers may be used to identify said progeny plant as a inbredsunflower SA425A progeny plant. Mean trait values may be used todetermine whether trait differences are significant, and preferably thetraits are measured on plants grown under the same environmentalconditions. Once such a variety is developed, its value is substantialsince it is important to advance the germplasm base as a whole in orderto maintain or improve traits such as yield, disease resistance, pestresistance, and plant performance in extreme environmental conditions.

Progeny of inbred sunflower SA425A may also be characterized throughtheir filial relationship with inbred sunflower SA425A, as for example,being within a certain number of breeding crosses of inbred sunflowerSA425A. A breeding cross is a cross made to introduce new genetics intothe progeny, and is distinguished from a cross, such as a self or a sibcross, made to select among existing genetic alleles. The lower thenumber of breeding crosses in the pedigree, the closer the relationshipbetween inbred sunflower SA425A and its progeny. For example, progenyproduced by the methods described herein may be within 1, 2, 3, 4, or 5breeding crosses of inbred sunflower SA425A.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cell tissue cultures from which sunflower plants canbe regenerated, plant calli, plant clumps, and plant cells that areintact in plants or parts of plants, such as leaves, pollen, embryos,cotyledons, hypocotyl, roots, root tips, anthers, pistils, flowers,ovules, seeds, stems, and the like.

The use of the terms “a,” “an,” and “the,” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. Forexample, if the range 10-15 is disclosed, then 11, 12, 13, and 14 arealso disclosed. All methods described herein can be performed in anysuitable order unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein, is intended merely to betterilluminate the invention and does not pose a limitation on the scope ofthe invention unless otherwise claimed. No language in the specificationshould be construed as indicating any non-claimed element as essentialto the practice of the invention.

Deposit Information

A deposit of the NUSEED AMERICAS INC. proprietary INBRED SUNFLOWERSA425A disclosed above and recited in the appended claims has been madewith the American Type Culture Collection (ATCC), 10801 UniversityBoulevard, Manassas, Va. 20110 under the terms of the Budapest Treaty.The date of deposit was Apr. 1, 2016. The deposit of 2,500 seeds wastaken from the same deposit maintained by NUSEED AMERICAS INC. sinceprior to the filing date of this application. All restrictions will beirrevocably removed upon granting of a patent, and the deposit isintended to meet all of the requirements of 37 C.F.R. §§1.801-1.809. TheATCC Accession Number is PTA-122987. The deposit will be maintained inthe depository for a period of thirty years, or five years after thelast request, or for the enforceable life of the patent, whichever islonger, and will be replaced as necessary during that period.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions, and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions, and sub-combinations as are within their truespirit and scope.

What is claimed is:
 1. A seed of inbred sunflower SA425A, wherein arepresentative sample of seed of said inbred sunflower was depositedunder ATCC Accession No. PTA-122987.
 2. A sunflower plant, or a partthereof, produced by growing the seed of claim
 1. 3. A sunflower plant,or a part thereof, having all of the physiological and morphologicalcharacteristics of the sunflower plant of claim
 2. 4. A tissue cultureproduced from protoplasts or cells from the plant of claim 2, whereinsaid cells or protoplasts are produced from a plant part selected fromthe group consisting of leaf, pollen, embryo, cotyledon, hypocotyl,meristematic cell root, root tip, pistil, anther, ovule, flower, shoot,stem, seed, and petiole.
 5. A sunflower plant regenerated from thetissue culture of claim 4, wherein the plant has all of themorphological and physiological characteristics of inbred sunflowerSA425A.
 6. A method for producing a hybrid sunflower seed, said methodcomprising crossing two sunflower plants and harvesting the resultantsunflower seed, wherein one sunflower plant is the sunflower plant ofclaim
 2. 7. A hybrid sunflower seed produced by the method of claim 6.8. A sunflower plant, or a part thereof, produced by growing said seedof claim
 7. 9. The method of claim 6, wherein one of said sunflowerplants is inbred sunflower SA425A and the other is transgenic.
 10. Amethod of producing a male sterile sunflower plant, wherein the methodcomprises introducing a nucleic acid molecule that confers malesterility into the sunflower plant of claim
 2. 11. A male sterilesunflower plant produced by the method of claim
 10. 12. A method ofproducing an herbicide resistant sunflower plant, wherein said methodcomprises introducing a gene conferring herbicide resistance into theplant of claim 2, wherein the gene is selected from the group consistingof glyphosate, sulfonylurea, imidazolinone, dicamba, glufosinate,phenoxy proprionic acid, L-phosphinothricin, cyclohexone,cyclohexanedione, triazine, and benzonitrile.
 13. An herbicide resistantsunflower plant produced by the method of claim
 12. 14. A method ofproducing a pest or insect resistant sunflower plant, wherein saidmethod comprises introducing a gene conferring pest or insect resistanceinto the plant of claim
 2. 15. A pest or insect resistant sunflowerplant produced by the method of claim
 14. 16. The sunflower plant ofclaim 15, wherein the gene encodes a Bacillus thuringiensis endotoxin.17. A method of producing a disease resistant sunflower plant, whereinsaid method comprises introducing a gene conferring disease resistanceinto the plant of claim
 2. 18. A disease resistant sunflower plantproduced by the method of claim
 17. 19. A method of producing asunflower plant wherein said method comprises introducing a transgeneinto the plant of claim
 2. 20. A sunflower plant produced by the methodof claim
 19. 21. A method of introducing a desired trait into inbredsunflower SA425A, wherein the method comprises: (a) crossing a plant ofinbred sunflower SA425A, wherein a representative sample of seed wasdeposited under ATCC Accession No. PTA-122987, with a plant of anothersunflower that comprises a desired trait to produce progeny plants, andwherein the desired trait is selected from the group consisting of malesterility, herbicide resistance, insect or pest resistance, modifiedbolting and resistance to bacterial disease, fungal disease or viraldisease; (b) selecting one or more progeny plants that have the desiredtrait; (c) backcrossing the selected progeny plants with inbredsunflower SA425A to produce backcross progeny plants; (d) selecting forbackcross progeny plants that have the desired trait; and (e) repeatingsteps (c) and (d) two or more times in succession to produce selectedthird or higher backcross progeny plants that comprise the desiredtrait.
 22. A sunflower plant produced by the method of claim 21, whereinthe plant has the desired trait and otherwise all of the physiologicaland morphological characteristics of inbred sunflower SA425A.